Methods and pharmaceutical compositions for the treatment of erythropoietic protoporphyria

ABSTRACT

The present invention relates to methods and pharmaceutical compositions for the treatment of Erythropoietic Protoporphyria. In particular, the present invention relates to a method for increasing the amount of functional FECH in a erythroid cell carrying the hypomorphic allele IVS3 48C/T (rs2272783) in trans to a deleterious mutation in the FECH gene comprising the step of consisting of bringing the erythroid cell into contact with at least one antisense oligonucleotide (ASO) comprising the sequence as set forth by SEQ ID NO: 2 (5′ gcagcctgagaaatgtttt 3′) to prevent splicing of the cryptic exon inserted into the mutant IVS3 48C/T (rs2272783) FECH mRNA.

FIELD OF THE INVENTION

The present invention relates to methods and pharmaceutical compositionsfor the treatment of Erythropoietic Protoporphyria.

BACKGROUND OF THE INVENTION

Erythropoietic Protoporphyria (EPP, MIM 177000) is a rare inheriteddisorder caused by the partial mitochondrial deficiency offerrochelatase (FECH, EC 4.99.1.1.), the last enzyme in the hemebiosynthesis pathway (Puy et al. 2010) (Balwani et al. 1993). FECH is aninner mitochondrial membrane enzyme that catalyzes the insertion of theferrous iron into free protoporphyrin IX (PPIX) to form heme. FECHdeficiency in bone marrow erythroid cells leads to the overproductionand accumulation of PPIX in the erythrocytes, and then to secondaryaccumulation of PPIX in the plasma, skin, bile and feces (Puy et al.2010). The commonest clinical manifestation is lifelong acutephotosensitivity of sun-exposed skin, first appearing in earlychildhood. Although it is generally a benign disease, hepaticcomplications such as cholelithiasis or, in about 2% of cases, cirrhosiswith rapidly fatal liver disease, may occur (Bloomer et al. 1998;Meerman 2000; Lyoumi et al. 2011). Cases of EPP have been reported inEurope, USA, China and Japan. So far, no case of EPP has been reportedin Black African subjects. Previously it was showed that the clinicaloutcome of EPP is due to the inheritance of a common hypomorphic allelein trans to a deleterious mutation; this reduces FECH activity below acritical 35% threshold of enzyme activity (Gouya et al. 1996; Gouya etal. 1999). A common intronic Single Nucleotide Polymorphism (SNP),IVS3-48C/T (rs2272783), is responsible for the low-expression of thehypomorphic IVS3-48C allele by modulating the use of a 3′ constitutivecryptic acceptor splice site located at the intron 3-exon 4 boundary,which leads to the pseudoexon inclusion of a portion of intron 3 (60%inclusion with the IVS3-48 C allele versus 20% with the T allele). Theaberrantly-spliced mRNA includes a premature stop codon, and is degradedby a nonsense-mediated mRNA decay mechanism (NMD) (Gouya et al. 2002).In overt cases, this low, steady-state mRNA level of the hypomorphicallele also results in FECH enzyme deficiency. The overall FECH activityfalls below a critical threshold of about 35% of normal, below whichPPIX accumulation and photosensitivity occur (Gouya et al. 2006; Taharaet al. 2010). Several studies in USA, Europe, and Asia have confirmedthat this mechanism is generally operative in EPP (Risheg et al. 2003;Wiman et al. 2003; Saruwatari et al. 2006; Kong et al. 2008; Whatley etal. 2010; Balwani et al. 2013). In France more than 90% of EPP patientsshow this striking inheritance pattern. Taken together, these findingssuggest that therapeutic benefits in EPP patients might be achieved byeven a modest increase in wild-type (WT) FECH protein. Thus, correctingthis single splicing mutation represents an attractive strategy thatcould improve the condition of the vast majority of EPP patients.Antisense oligonucleotides (ASOs), which are generally used to inhibitgene expression, can also be used to modulate pre-mRNA splicing bytargeting splice sites, or positive or negative elements that affectsplice-site selection (Kole et al. 2012).

SUMMARY OF THE INVENTION

The present invention relates to methods and pharmaceutical compositionsfor the treatment of Erythropoietic Protoporphyria.

DETAILED DESCRIPTION OF THE INVENTION

In 90% of erythropoietic protoporphyria (EPP) patients, the diseaseresults from the inheritance of a common hypomorphic FECH allele intrans to a deleterious FECH mutation. The activity of the resulting FECHenzyme falls below the critical threshold of 35% leading to theaccumulation of free portoporphyrin IX (PPIX) in bone marrowerythroblasts and in red cells. The mechanism of low expression involvesa biallelic polymorphism (IVS3-48C/T) localized in intron 3. TheIVS3-48C allele increases usage of the exon 3-4 3′ cryptic splice site,resulting in the transcription of an unstable mRNA with a premature STOPcodon, reducing the abundance of wild-type FECH mRNA and finallyreducing FECH activity. Through a candidate sequence approach and anantisense oligonucleotide-tiling method, the inventors identified asequence which when targeted by an antisense oligonucleotide (ASO-V1)prevented usage of the cryptic splice site. In lymphoblastoid cell linesderived from symptomatic EPP patients, transfection of ASO-V1 reducedthe usage of the cryptic splice site, and efficiently redirected thesplicing of intron 3 towards the physiological acceptor site, therebyincreasing the amount of functional FECH mRNA. Finally, theadministration of ASO-V1 into human developing erythroblasts from anovertly EPP patient markedly increased the production of WT FECH mRNA,and reduced the accumulation of PPIX to a level similar to that measuredin asymptomatic EPP patients. Thus, EPP appears to be a prototypicMendelian disease where the in-vivo correction of a single splicingdefect is likely to improve the condition of the vast majority ofovertly EPP patients worldwide. The invention thus provides use of suchexon-skipping strategy for the treatment of ErythropoieticProtoporphyria.

The present invention relates to a method for increasing the amount offunctional FECH in a erythroid cell carrying the hypomorphic allele IVS348C/T (rs2272783) in trans to a deleterious mutation in the FECH genecomprising the step of consisting of bringing the erythroid cell intocontact with at least one antisense oligonucleotide (ASO) comprising thesequence as set forth by SEQ ID NO: 2 (5′ gcagcctgagaaatgtttt 3′) toprevent splicing of the cryptic exon inserted into the mutant IVS3 48C/T(rs2272783) FECH mRNA.

The term “FECH” has its general meaning in the art and refers to theferrochelatase (FECH, EC 4.99.1.1.), which is the last enzyme in theheme biosynthesis pathway (Puy et al. 2010) (Balwani et al. 1993). FECHis an inner mitochondrial membrane enzyme that catalyzes the insertionof the ferrous iron into free protoporphyrin IX (PPIX) to form heme. Anexemplary native human nucleotide sequence encoding for FECH is providedin GenBank with the access number NM_000140.3 (SEQ ID NO:1).

As used, the expression “deleterious mutation in the FECH gene” refersto any mutation which results in to a dysfunction of the FECH proteinleading to the loss of its activity. Deleterious mutations in the FECHgene have fully been exemplified in the prior art and thus the skilledman in the art can easily understand such a mutation (as exemplified bythe database found at www.hgmd.cf.ac.uk/ac/index.php).

ASOs employed in the practice of the invention are generally from about19 to about 30 nucleotides in length, and may be for example, about 19,or about 20, about 25 or about 30 nucleotides or more in length.

In a particular embodiment, the ASO comprises or consists of a nucleicacid sequence selected from Table A.

TABLE A position sequence -43 -63 tagcagcctgagaaatgtttt SEQ ID NO: 3-44 -63 agcagcctgagaaatgtttt SEQ ID NO: 4 -45 -63 gcagcctgagaaatgttttSEQ ID NO: 2 -43 -64 tagcagcctgagaaatgttttct SEQ ID NO: 5 -45 -64gcagcctgagaaatgttttc SEQ ID NO: 6 -44 -64 agcagcctgagaaatgttttcSEQ ID NO: 7 -43 -65 agcagcctgagaaatgttttct SEQ ID NO: 8 -44 -65agcagectgagaaatgttttc SEQ ID NO: 9 -45 -65 gcagcctgagaaatgttttctSEQ ID NO: 10

The ASO used in the practice of the invention may be of any suitabletype. The one skilled in the art can easily provide some modificationsthat will improve the clinical efficacy of the ASO (C. Frank Bennett andEric E. Swayze, RNA Targeting Therapeutics: Molecular Mechanisms ofAntisense Oligonucleotides as a Therapeutic PlatformAnnu. Rev.Pharmacol. Toxicol. 2010.50:259-293.). Typically, chemical modificationsinclude backbone modifications, heterocycle modifications, sugarmodifications, and conjugations strategies. For example the ASO may beselected from the group consisting of oligodeoxyribonucleotides,oligoribonucleotides, LNA, oligonucleotide, morpholinos,tricyclo-DNA-antisense oligonucleotides, U7- or U1-mediated ASOs orconjugate products thereof such as peptide-conjugated ornanoparticle-complexed ASOs. Indeed, for use in vivo, the ASO may bestabilized. A “stabilized” ASO refers to an ASO that is relativelyresistant to in vivo degradation (e.g. via an exo- or endo-nuclease).Stabilization can be a function of length or secondary structure. Inparticular, ASO stabilization can be accomplished via phosphate backbonemodifications.

In a particular embodiment, the ASO according to the invention is a LNAoligonucleotide. As used herein, the term “LNA” (Locked Nucleic Acid)(or “LNA oligonucleotide”) refers to an oligonucleotide containing oneor more bicyclic, tricyclic or polycyclic nucleoside analogues alsoreferred to as LNA nucleotides and LNA analogue nucleotides. LNAoligonucleotides, LNA nucleotides and LNA analogue nucleotides aregenerally described in International Publication No. WO 99/14226 andsubsequent applications; International Publication Nos. WO 00/56746, WO00/56748, WO 00/66604, WO 01/25248, WO 02/28875, WO 02/094250, WO03/006475; U.S. Pat. Nos. 6,043,060, 6,268,490, 6,770,748, 6,639,051,and U.S. Publication Nos. 2002/0125241, 2003/0105309, 2003/0125241,2002/0147332, 2004/0244840 and 2005/0203042, all of which areincorporated herein by reference. LNA oligonucleotides and LNA analogueoligonucleotides are commercially available from, for example, ProligoLLC, 6200 Lookout Road, Boulder, Colo. 80301 USA. In a particularembodiment, the ASO comprise the 5′ gcEgcLtgEgaEatPttZt 3′ sequence asdescribed in Table 1.

Other possible stabilizing modifications include phosphodiestermodifications, combinations of phosphodiester and phosphorothioatemodifications, methylphosphonate, methylphosphorothioate,phosphorodithioate, p-ethoxy, and combinations thereof. Chemicallystabilized, modified versions of the ASO also include “Morpholinos”(phosphorodiamidate morpholino oligomers, PMOs), 2′-O-Met oligomers,tricyclo (tc)-DNAs, U7 short nuclear (sn) RNAs, ortricyclo-DNA-oligoantisense molecules (U.S. Provisional PatentApplication Ser. No. 61/212,384 For: Tricyclo-DNA AntisenseOligonucleotides, Compositions and Methods for the Treatment of Disease,filed Apr. 10, 2009, the complete contents of which is herebyincorporated by reference).

Other forms of ASOs that may be used to this effect are ASO sequencescoupled to small nuclear RNA molecules such as U1 or U7 in combinationwith a viral transfer method based on, but not limited to, lentivirus oradeno-associated virus (Denti, M A, et al, 2008; Goyenvalle, A, et al,2004).

For use in the instant invention, the ASOs of the invention can besynthesized de novo using any of a number of procedures well known inthe art. For example, the b-cyanoethyl phosphoramidite method (Beaucageet al., 1981); nucleoside H-phosphonate method (Garegg et al., 1986;Froehler et al., 1986, Garegg et al., 1986, Gaffney et al., 1988). Thesechemistries can be performed by a variety of automated nucleic acidsynthesizers available in the market. These nucleic acids may bereferred to as synthetic nucleic acids. Alternatively, ASO can beproduced on a large scale in plasmids (see Sambrook, et al., 1989). ASOcan be prepared from existing nucleic acid sequences using knowntechniques, such as those employing restriction enzymes, exonucleases orendonucleases. ASO prepared in this manner may be referred to asisolated nucleic acids.

In a particular embodiment, two or even more ASOs can also be used atthe same time; this may be particularly interesting when the ASO arevectorized within an expression cassette (as for example by U7 or U1cassettes).

In a particular embodiment, the ASO of the present invention isconjugated to a second molecule. Typically said second molecule isselected from the group consisting of aptamers, antibodies orpolypeptides. For example, the ASO of the present invention may beconjugated to a cell penetrating peptide. Cell penetrating peptides arewell known in the art and include for example the TAT peptide. In aparticular embodiment, the second molecule is able to target theerythroid cell. In a particular embodiment, the molecule targets thetransferrin receptor 1 (i.e. CD71). Several peptides and aptamers thatbind with high affinity to human CD71 and display endocytotic propertiesare described in Lee J H, Engler J A, Collawn J F, Moore B A (2001)Receptor mediated uptake of peptides that bind the human transferrinreceptor. Eur J Biochem 268:2004-2012 and in Wilner S E, Wengerter B,Maier K, de Lourdes Borba Magalhaes M, Del Amo D S, Pai S, Opazo F,Rizzoli S O, Yan A, Levy M (2012) An RNA alternative to humantransferrin: a new tool for targeting human cells. Mol Ther NucleicAcids 1:e21.

The method of the invention is particularly suitable for the treatmentof Erythropoietic Protoporphyria in a patient harbouring the commonhypomorphic allele IVS3 48C/T (rs2272783) in trans to a deleteriousmutation in the FECH gene. Accordingly, the present invention relates toan ASO as described above for use in the treatment of ErythropoieticProtoporphyria.

In a particular embodiment; antisense oligonucleotides of the inventionmay be delivered in vivo alone or in association with a vector. In itsbroadest sense, a “vector” is any vehicle capable of facilitating thetransfer of the antisense oligonucleotide of the invention to the cells.Preferably, the vector transports the nucleic acid to cells with reduceddegradation relative to the extent of degradation that would result inthe absence of the vector. In general, the vectors useful in theinvention include, but are not limited to, naked plasmids, non viraldelivery systems (electroporation, sonoporation, cationic transfectionagents, liposomes, etc. . . . ), phagemids, viruses, other vehiclesderived from viral or bacterial sources that have been manipulated bythe insertion or incorporation of the antisense oligonucleotide nucleicacid sequences. Viral vectors are a preferred type of vector andinclude, but are not limited to nucleic acid sequences from thefollowing viruses: RNA viruses such as a retrovirus (as for examplemoloney murine leukemia virus and lentiviral derived vectors), harveymurine sarcoma virus, murine mammary tumor virus, and rous sarcomavirus; adenovirus, adeno-associated virus; SV40-type viruses; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vacciniavirus; polio virus. One can readily employ other vectors not named butknown to the art. In a preferred embodiment, the antisenseoligonucleotide nucleic acid sequence is under the control of aheterologous regulatory region, e.g., a heterologous promoter. Thepromoter can also be, e.g., a viral promoter, such as CMV promoter orany synthetic promoters.

The present invention also provides a pharmaceutical compositioncontaining an antisense oligonucleotide of the invention (or a vector ofthe invention) for the treatment of Erythropoietic Protoporphyria in apatient harbouring the common hypomorphic allele IVS3 48C/T (rs2272783)in trans to a deleterious mutation in the FECH gene.

Typically, pharmaceutical compositions of the present invention includea pharmaceutically or physiologically acceptable carrier such as saline,sodium phosphate, etc. The compositions will generally be in the form ofa liquid, although this need not always be the case. Suitable carriers,excipients and diluents include lactose, dextrose, sucrose, sorbitol,mannitol, starches, gum acacia, calcium phosphates, alginate,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, celluose, water syrup, methyl cellulose, methyland propylhydroxybenzoates, mineral oil, etc. The formulations can alsoinclude lubricating agents, wetting agents, emulsifying agents,preservatives, buffering agents, etc. Those of skill in the art willalso recognize that nucleic acids are often delivered in conjunctionwith lipids (e.g. cationic lipids or neutral lipids, or mixtures ofthese), frequently in the form of liposomes or other suitable micro- ornano-structured material (e.g. micelles, lipocomplexes, dendrimers,emulsions, cubic phases, etc.).

One skilled in the art will recognize that the amount of an ASO to beadministered will be an amount that is sufficient to induce ameliorationof unwanted disease symptoms. Such an amount may vary inter aliadepending on such factors as the gender, age, weight, overall physicalcondition, of the patient, etc. and may be determined on a case by casebasis. The amount may also vary according to the type of condition beingtreated, and the other components of a treatment protocol (e.g.administration of other medicaments such as steroids, etc.). If aviral-based delivery of ASOs is chosen, suitable doses will depend ondifferent factors such as the viral strain that is employed, the routeof delivery (intramuscular, intravenous, intra-arterial or other). Thoseof skill in the art will recognize that such parameters are normallyworked out during clinical trials. Further, those of skill in the artwill recognize that, while disease symptoms may be completely alleviatedby the treatments described herein, this need not be the case. Even apartial or intermittent relief of symptoms may be of great benefit tothe recipient. In addition, treatment of the patient is usually not asingle event. Rather, the ASOs of the invention will likely beadministered on multiple occasions, that may be, depending on theresults obtained, several days apart, several weeks apart, or severalmonths apart, or even several years apart. This is especially true wherethe treatment of Erythropoietic Protoporphyria is concerned since thedisease is not cured by this treatment, i.e. the gene that encodes theprotein will still be defective and the encoded protein will stillpossess an unwanted, destabilizing feature such as an exposedproteolytic recognition site, unless the ASOs of the invention areadministered.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of exon 3-4 FECH gene splicing.

FIG. 1A: The IVS3-48T/C transition modulates the splicing efficiency ofa constitutive cryptic acceptor splice site. −63 bp: position of thecryptic acceptor splice site. Sequence shown is that of nucleotides51-94 of SEQ ID NO:58.

FIG. 1B: Position of the LNA-ASOs targeting the putative cryptic branchpoint (−97-116), the cryptic acceptor splice site (−54-74), and both thecryptic acceptor splice site and the IVS3-48 locus (−45-63). Sequenceshown is that of nucleotides 11-94 of SEQ ID NO:58.

Putative cryptic branch point.

FIG. 1C: Inhibition of abnormal FECH splicing by 3 LNAs-ASO.

Cos 7 cells were transiently cotransfected with the FECH-C-pcDNA3minigene and 50, 125, or 250 nM of the appropriate LNA oligonucleotides(Eurogentec, Angers, France) using lipofectamine 2000 reagent (Lifetechnologies, Saint-Aubin, France). RNA was extracted 24 h aftertransfection with RNA plus reagent (MP Biomedical, Illkirch, France). RTPCR products are analyzed on 3% agarose gel. The migration positions ofthe normal exon 3-4 81-bp amplimer and the aberrantly-spliced 144-bpproduct are indicated on the right. PCR primers were selected to behuman specific. Ratios of the aberrantly-spliced RNA to the total RNAare indicated at the bottom.

FIG. 2: Restoration of WT FECH mRNA production in the LBCLs of EPPpatients.

FIG. 2A: Pedigree of the EPP family used. “M”: c. 709delT FECH genemutation. “T”: IVS3-48T allele. “C”: IVS3-48C allele. Subjects I1 andII3 are asymptomatic carriers of the c. 709delT mutation. Subjects II1and II2 are overt EPP patients. FC: FECH activity in nmoles ofZn-Mesoporphyrin/mg of protein/hour.

FIG. 2B: The LBCLs were transfected with 125 nM of −45-63 or mock(−43-65 sense sequence) LNA-ASOs and emetin was added 24 hours later.Total RNA was extracted 48 h after transfection. The migration positionsof the normal exon 3-4 126-bp amplimer, and of the aberrantly spliced192-bp product are indicated on the right. This figure represents oneexample of 6 experiments.

FIG. 2C: The ratios of the aberrantly-spliced RNA to the total RNA arepresented as box plots showing the median, the quartiles, the 90th andthe 10th percentiles. n=6 experiments. The Mann-Whitney statistical testwas used with Prism 4 software (GraphPad software, La Jolla, USA).

FIG. 2D: Total RNA was extracted 24 h and 48 h after transfection withthe −45-63 or mock LNA-ASOs without emetin. WT FECH RNA was analyzed byRT-qPCR with specific primers of the normal exon 3-4 spliced RNA andnormalized with two genes (B2M and HPRT1). n=6 independent experiments.The Mann-Whitney statistical test was used with Prism 4 software.

FIG. 3: In vitro erythroid differentiation of CD34⁺ cells isolated fromperipheral whole blood in the presence of 45 μM, FITC-labeled V1 or mockmorpholinos.

Total mononuclear cells were isolated by Ficoll density-gradientcentrifugation (LSM; PAA laboratories, Velizy-Villacoublay, France),from 60 ml peripheral whole blood. CD34⁺ cells were purified usingimmunomagnetic beads (MACS CD34 MicroBead Kit; Miltenyi Biotec, Paris,France). For the erythroid differentiation, CD34.sup.+ cells were grownin Iscove modified Dulbecco medium (Invitrogen, Saint Aubin, France)supplemented with 15% BIT 9500 (StemCell Technologies, Grenoble,France), 2 IU/mL EPO, 100 ng/mL SCF, 10 ng/mL IL-6 and 10 ng/mL IL-3(Miltenyi Biotec).

Time kinetic analysis of PPIX accumulation was determined by flowcytometry at four stages of differentiation—CFU E, Pro E, Int E, andLate E. Indicated values in the graphs represent ratios between the MeanFluorescence Intensity (MFI) values of accumulated PPIX and MFI ofFITC-positive cells. (A) Normalized MFI of PPIX in erythroid cells fromovert EPP patient A and control patient C, treated either with PMO-V1(▴) or with PMO-mock (▪). (B) Normalized MFI of PPIX in erythroid cellsfrom overt EPP patient B, and asymptomatic carrier D, treated eitherwith PMO-V1 (▴) or with PMO-mock (▪).

FIG. 4: Erythrocyte protoporphyrin IX in 40 EPP families.

Total erythrocyte PPIX was measured in 40 EPP families each having oneovert patient, one asymptomatic patient, and one healthy subject. Theresults were expressed as a histogram showing the mean and the standarddeviation. Means were compared using “t” test with Prism 4 software.

FIG. 5: ASO-tiling approach to search for regulating cis-acting elementsof exon 3-4 splicing residing within the 130-bp stretch upstream of exon4.

FIG. 5A: Schematic representation of the binding site of the 13 ASOsused. Sequence shown is that of SEQ ID NO:58.

FIG. 5B: Cos 7 cells were transiently cotransfected with theFECH-C-pcDNA3 minigene and 50, 125, or 250 nM of the appropriate LNA-ASOusing lipofectamine 2000 reagent. Total RNA was extracted 24 h aftertransfection. RT-PCR products were analyzed on 3% agarose gel. Themigration positions of the normal exon 3-4 81-bp amplimer, and theaberrantly-spliced 144-bp product are indicated on the right. PCRprimers had been chosen to be human specific. Ratios of theaberrantly-spliced RNA to the total RNA are indicated at the bottom.

FIG. 6: Microwalk around position −45 in FECH intron 3

FIG. 6A: Schematic representation of the binding site of the 18LNA-ASOs. Sequence shown is that of nucleotides 54-135 of SEQ ID NO:58.

FIG. 6B and FIG. 6C: Cos 7 cells were transiently cotransfected with theFECH-C-pcDNA3 minigene, and 50, 125, or 250 nM of the appropriateLNA-ASO using lipofectamine 2000 reagent. Total RNA was extracted 24 hafter transfection. RT-PCR products were analyzed on 3% agarose gel. Themigration positions of the normal exon 3-4 81-bp amplimer, and theaberrantly-spliced 144-bp product are indicated on the right. Ratios ofthe aberrantly-spliced RNA to the total RNA are indicated at the bottom.

FIG. 6B: 5′microwalk from position −45.

FIG. 6C: 3′microwalk from position −45

FIG. 7: Microwalk around position −45-63

FIG. 7A: Schematic representation of the binding site of the 8 LNA-ASOs.Sequence shown is that of nucleotides 54-135 of SEQ ID NO:58.

FIG. 7B: Cos 7 cells were transiently cotransfected with theFECH-C-pcDNA3 minigene and 50, 125, or 250 nM of the appropriate LNA-ASOusing lipofectamine 2000 reagent. Total RNA was extracted 24 h aftertransfection. RT-PCR products were analyzed on 3% agarose gel. Themigration positions of the normal exon 3-4 81-bp amplimer, and of theaberrantly-spliced 144-bp product are indicated on the right. Ratios ofthe aberrantly-spliced RNA to the total RNA are indicated at the bottom.

FIG. 8: WT FECH gene expression during in vitro erythroiddifferentiation of CD34⁺ cells isolated from the peripheral whole bloodof a normal subject. WT FECH RNA was analyzed by RT-qPCR with specificprimers of the normal exon 3-4 spliced RNA and normalized using twogenes (B2M and HPRT1).

FIG. 9: Cytospin preparations of cells stained with May-Grunwald Giemsaillustrate the morphologic changes.

Four populations of erythroid cells were distinguished—corresponding tostages characterized by the predominance of CFU E, pro-erythroblasts(Pro E), basophilic and polychromatophilic stage (Int E), andreticulocytes (Late E). Representative pictures were taken at ×40magnification. “Control”: normal subject C; “EPP”: overt EPP patient A.

EXAMPLE 1: ANTISENSE OLIGONUCLEOTIDE-BASED THERAPY IN HUMANERYTHROPOIETIC PROTOPORPHYRIA

Erythropoietic Protoporphyria (EPP, MIM 177000) is a rare inheriteddisorder caused by the partial mitochondrial deficiency offerrochelatase (FECH, EC 4.99.1.1.), the last enzyme in the hemebiosynthesis pathway (Puy et al. 2010) (Balwani et al. 1993). FECH is aninner mitochondrial membrane enzyme that catalyzes the insertion of theferrous iron into free protoporphyrin IX (PPIX) to form heme. FECHdeficiency in bone marrow erythroid cells leads to the overproductionand accumulation of PPIX in the erythrocytes, and then to secondaryaccumulation of PPIX in the plasma, skin, bile and feces (Puy et al.2010). The commonest clinical manifestation is lifelong acutephotosensitivity of sun-exposed skin, first appearing in earlychildhood. Although it is generally a benign disease, hepaticcomplications such as cholelithiasis or, in about 2% of cases, cirrhosiswith rapidly fatal liver disease, may occur (Bloomer et al. 1998;Meerman 2000; Lyoumi et al. 2011).

Cases of EPP have been reported in Europe, USA, China and Japan. So far,no case of EPP has been reported in Black African subjects. Previouslywe showed that the clinical outcome of EPP is due to the inheritance ofa common hypomorphic allele in trans to a deleterious mutation; thisreduces FECH activity below a critical 35% threshold of enzyme activity(Gouya et al. 1996; Gouya et al. 1999). A common intronic SingleNucleotide Polymorphism (SNP), IVS3-48C/T (rs2272783, FIG. 1A), isresponsible for the low-expression of the hypomorphic IVS3-48C allele bymodulating the use of a 3′ constitutive cryptic acceptor splice sitelocated at the intron 3-exon 4 boundary, which leads to the pseudoexoninclusion of a portion of intron 3 (60% inclusion with the IVS3-48 Callele versus 20% with the T allele). The aberrantly-spliced mRNAincludes a premature stop codon, and is degraded by a nonsense-mediatedmRNA decay mechanism (NMD) (Gouya et al. 2002). In overt cases, thislow, steady-state mRNA level of the hypomorphic allele also results inFECH enzyme deficiency. The overall FECH activity falls below a criticalthreshold of about 35% of normal, below which PPIX accumulation andphotosensitivity occur (Gouya et al. 2006; Tahara et al. 2010). Severalstudies in USA, Europe, and Asia have confirmed that this mechanism isgenerally operative in EPP (Risheg et al. 2003; Wiman et al. 2003;Saruwatari et al. 2006; Kong et al. 2008; Whatley et al. 2010; Balwaniet al. 2013). In France more than 90% of EPP patients show this strikinginheritance pattern.

Taken together, these findings suggest that therapeutic benefits in EPPpatients might be achieved by even a modest increase in wild-type (WT)FECH protein. Thus, correcting this single splicing mutation is anattractive strategy that could improve the condition of the vastmajority of EPP patients. Antisense oligonucleotides (ASOs), which aregenerally used to inhibit gene expression, can also be used to modulatepre-mRNA splicing by targeting splice sites, or positive or negativeelements that affect splice-site selection (Kole et al. 2012).

In this study, we applied two strategies to identifying ASOs thatrepress the partial inclusion of intron 3 in FECHmRNA: firstly anASO-tiling method to search for regulating cis-acting elements residingwithin the 130-bp stretch upstream of exon 4, and secondly an orientatedstrategy using three ASOs targeting the putative IVS3-48C-activatedcryptic branch point, the cryptic acceptor splice site, or both thecryptic splice site and the IVS3-48C nucleotide (FIG. 1B and Table 1).For all these experiments, we used 15-mer Locked Nucleic Acids (LNAs)that demonstrate nuclease resistance, and enhance affinity forhybridization to complementary RNA (Kurreck et al. 2002). This class ofASO is highly effective for modifying pre-mRNA splicing patterns(Roberts et al. 2006). To investigate the effects of individual ASOs onsplicing, we constructed two FECH minigenes (FECH-C-pcDNA3 andFECH-T-pcDNA3) comprising exon 3 (121 bp), a shortened intron 3 (300 bp,with either the IVS3-48C or the IVS3-48T genotype) and exon 4 (193 bp).They were transfected into Cos 7 cells using lipofectamine reagent(Invitrogen, Lyon, France). Two days after transfection, exon 3-4 FECHmRNA was analyzed by RT-PCR, using a pair of human primers (Table 4).These primers did not amplify the endogenous Cos 7 FECH transcript (FIG.1C). The extent of intron-3 inclusion was calculated as the ratiobetween the density of the abnormally-spliced FECH mRNA to that of thenormally- plus abnormally-spliced mRNA. FECH minigene transfection inCos 7 cells recapitulated the splicing pattern as shown inlymphoblastoid cell lines (LBCL) of IVS3-48C/C or T/T patients (Gouya etal. 2002). The FECH-C-pcDNA3 minigene revealed a ratio of about 60%, andthe FECH-T-pcDNA3 minigene one of 20%. For the ASO-tiling experiments,we cotransfected individually the FECH-C-pcDNA3 minigene with each ofthe ASOs (FIG. 5A and Table 4; 13 ASOs, overlapping by 5 nt). None ofthe 13 LNA-ASOs used in the initial “walk” along intron 3 reduced intron3 retention (FIG. 5B). Only ASO −45-63, obtained using the orientatedstrategy, complementary to a sequence including the IVS3-48 polymorphismand the 3′ cryptic acceptor splice site, reduced intron 3 inclusion toabout 20% of the total RNA, a level similar to the level obtained withthe FECH-T-pcDNA3 construction (FIG. 1C). A dose-response effect showedthat this effect was already evident at 50 nM ASO concentration, and wasmaximal at 125 nM (FIG. 1C). To further refine the targeting sequence,we microwalked around the −45 position using 18 additional 15 merLNA-ASOs (FIG. 6A and Table 5). Again, none displayed any effect,demonstrating that both the IVS3-48 locus and the −63 acceptor splicesite have to be targeted to achieve the repression of intron 3 inclusion(FIG. 6B). Finally, we tested whether the splicing was sensitive to thelength of the ASO. We used eight additional LNA-ASOs, which were one ortwo bases longer than LNA-ASO −45-63 at either one or both extremities(FIG. 6A and Table 4). All these LNA-ASOs reduced intron 3 inclusion,but none was more effective than the −45-63 LNA-ASO (FIG. 7B).

To further investigate whether by reducing intron 3 retention, we alsoinduced a higher WT intron 3-exon 4 splicing rate, we measured splicingin the LBCL of a sib-pair of EPP patients (FIG. 2B). The FECH genotypeof both patients included the c.709delT mutation, which was trans to theIVS3-48C allele in the overtly EPP patient (subject II1, showingclassical symptoms of EPP), and trans to a T allele in the asymptomaticEPP patient (FIG. 2A, subject II3, without any symptoms of EPP). Theoptimal concentration (125 nM) of −45-63 LNA-ASO or mock-ASO (acomplementary sequence of the −45-63 ASO) was transfected usinglipofectamine reagent. Three hours after transfection, emetine(Sigma-Aldrich, Saint-Quentin Fallavier, France) was added to the mediumto block NMD. Forty-eight hours after ASO transfection, endogenous exon3-4 splicing was analyzed using exon 3-4 RT-PCR (Table 4; FIG. 2B). Asexpected, intron 3 retention in the LBCL of patient II3 (transfectedwith the mock ASO) was about 25% lower than in the LBLCL of patient II1(abnormal mRNA/total mRNA ratio 0.30 versus 0.38, p=0.0022; FIG. 2C).Interestingly, when the LBCL of patient II1 was transfected with the−45-63 LNA-ASO, the ratio fell to a level almost identical to thatmeasured in the asymptomatic II3 patient (0.32 versus 0.30, notsignificant; FIG. 2C). WT FECH mRNA was then measured by RT-qPCR usingprimers specific to the WT exon 3-4 boundary (Table 4). Twenty-fourhours after ASO transfection, no differences were observed between themock and −45-63 LNA-ASO-transfected cells (FIG. 2D). As expected, WTFECH mRNA was 30% more abundant in LBCLs of the asymptomatic versussymptomatic patient. Forty-eight hours after transfection with the−45-63 LNA-ASO, LBCL of the overt II1 patient showed a considerableincrease in WT FECH mRNA, which was 1.8 and 1.7 times higher than theII1 mock transfected cells and the II3−45-63 LNA-ASO transfected cells,respectively (FIG. 2D).

In summary, we identified a −45-63 nt sequence which when targeted byASO reduced intron 3 inclusion in LBCL with a IVS3-48T/C genotype to alevel comparable to that of the IVS3-48T/T genotype, and increased WTmRNA production in the cells of an overt patient to a higher level thanthat measured in an asymptomatic EPP patient. Taken together, theseresults suggest that the −45-63 ASO has considerable therapeuticpotential. The −45-63 sequence is intronic with regard to physiologicalexon 3-4 splicing, but becomes exonic when the 63 cryptic splice site isused. The mechanisms underlying splicing redirection from the 3-4boundary of the cryptic to the physiological exon are complex, and notyet fully understood. Blocking exclusively either the 3′ aberrant splicesite (6 ASOs: −50-64; −51-65; −52-66; −53-67; −61-75 and −54-74) or theIVS3-48 locus (11 ASOs: −34-48 to −39-53; −41-65 and −45-59 to −48-62)was not sufficient to restore proper splicing. In the competitionbetween the cryptic and the physiological splice sites, redirection ofsplicing toward the physiological site has to include blocking both thecryptic splice site and the IVS3-48 locus, suggesting that this regionmay include an exonic splicing enhancer (ESE) of cryptic splicing or anintronic splicing inhibitor (ISI) of exon 3-4 splicing.

Bone marrow erythroblasts are the primary source of excessive PPIXproduction in EPP; secondarily this leads to its accumulation in othertissues. This means that these cells are the relevant tissue to betargeted by a therapeutic approach. This prompted us to test the effectof the −45-63 nt ASO (referred to as V1 hereafter) on erythroidprecursor cells from overt and asymptomatic EPP patients.

We cultured CD34⁺-derived erythroid progenitors from two overt EPPpatients (patients A and B), one asymptomatic patient (patient D), andone control subject (subject C). Overt patients A and B both had theclassical history of skin photosensitivity beginning during childhood, ahigh level of erythrocyte free protoporphyrin, and 25-30% residual FECHactivity in peripheral blood mononuclear cells. Their FECH genotypeconsisted of a deleterious FECH mutation in trans to the hypomorphicIVS3-48C allele (Table 2). The nonsense mutation of patient A introduceda premature stop codon probably associated with mRNA degradation, andthe mutation in patient B was responsible for aberrant exon 10 splicingconserving the correct reading frame. The third patient (patient D) wasan asymptomatic EPP patient with a homozygous IVS3-48T/T genotype. Hererythrocyte free protoporphyrins were slightly above the normal limit(×2), and her FECH activity was about 50% below normal in peripheralblood mononuclear cells (Table 2). Her daughter is an overt EPP patient,with a classical history of cutaneous photosensitivity, a high level offree PPIX accumulation in erythrocytes (65 μmol/L RBC), a morepronounced FECH deficiency in lymphocytes than her mother (1.2 versus1.8 for her mother), and an IVS3-48C allele inherited from the healthyfather (Table 2). Our study was conducted in accordance with the WorldMedical Association Declaration of Helsinki ethical principles formedical research involving human subjects, and its subsequentamendments. All patients gave informed consent before undergoinginvestigation.

The phenotype differentiation of erythroid cells was monitored by flowcytometry (FACS Canto II, BD Biosciences, Le Pont de Claix, France).Cells were stained with fluorescently-labeled antibodies against CD34-PE(A07776, Beckman Coulter, Roissy CDG, France), CD36-FITC (IM0766U,Beckman Coulter), CD71-FITC (IM0483, Beckman Coulter), and GPA-PE(MHGLA04, Invitrogen). Hemoglobinisation was monitored daily bybenzidine staining. Cell morphology was established afterMay-Grünwald-Giemsa staining of cytospin preparations using lightmicroscopy.

For the oligonucleotide treatment, liposomal transfections of LNA-ASO V1were ineffective due to their high toxicity and poor transfectionefficiency (data not shown). We therefore used a free uptake method byadding a morpholino-ASO to the culture medium at a final concentrationof 45 μM (Sazani et al. 2001). The original method was developed by R.Kole to direct morpholino-ASOs to erythroid precursors in order torestore human beta globin gene expression in Human IVS2-654 thalassemicerythroid cells (Suwanmanee et al. 2002). The authors also showed thatadding labelled morpholinos to the culture medium from day 8 to 17resulted in strong nuclear staining. The morpholinos were prepared andpurified by Gene Tools, LLC (Philomath, USA). Two morpholino-ASOs wereused, targeting the −45-63 sequence in either the antisense (PMO-V1) orthe complementary sequence for the mock oligonucleotide as control(PMO-mock), respectively. The oligonucleotides were labelled usingfluorescein isithiocyanate (FITC).

The effects on splicing and on PPIX overproduction were analyzedsequentially during the differentiation of the erythroid progenitors.Due to the limited number of cells available, FECH activity could not beused for these experiments. WT FECH mRNA and ALAS2 mRNA were quantifiedby RT-qPCR (Table 4). Protoporphyrin overproduction was analyzed by flowcytometry (excitation at 405 nm, emission at 660 nm, Canto II, BectonDickinson France, Rungis) at four time points during the erythroidculture, and the results were normalized by FITC fluorescence to allowfor the exact amount of ASO taken in by the cells (FIG. 3).

These points were established on the basis of the expression of surfacemarkers (CD71, GPA), corresponding to the major stages of development:CFU E (Colony unit etc. CD71^(low), GPA⁻), pro-erythroblasts (Pro E;CD71^(high), GPA^(−/low)), intermediate (Int E; CD71^(high), GPA^(high))and late (Late E; CD71^(low), GPA^(high)) erythroblasts. To determinethe optimal conditions for antisense oligonucleotide treatments, aculture of CD34⁺ mononuclear cells isolated from whole peripheral bloodfrom a healthy control was established. The time course of FECH mRNAexpression was assayed by RT-qPCR. FECH mRNA increased dramatically fromthe CFU E stage to the Late E stage, with a 23 fold induction (FIG. 8).Parallel erythroid differentiation was confirmed on the basis of thecell morphology (FIG. 9), and by the progressive appearance ofhemoglobinized cells ranging from 0% hemoglobinization at the CFU Estage to >95% at the Late E stage. Morpholino oligonucleotides wereadded before inducing heme production at the CFU E stage (afterapproximately 7 days in culture), and were maintained at the sameconcentration until mature cells were obtained.

Two independent experiments were set up, one using cells from subjects Aand C, and the other with those from subjects B and D (FIG. 3). WT FECHmRNA was quantified by RT-qPCR at the late E stage, when the preliminaryexperiment had shown that FECH mRNA expression was highest (FIG. 8). Atthis stage of differentiation, antisense ASO-treated cells from patientsA and B showed increases of 58% and 98%, respectively, in WT FECH mRNAas compared to the mock treated cells (Table 3). In the firstexperiment, FECH mRNA in V1 treated cells of patient A remained 50%lower than in control subject C. This could have been due to theseverity of the mutations: a one base pair deletion introducing apremature STOP codon for patient A, potentially leading to a nullallele. The improvement in FECH gene expression could be attributedmainly to the redirection of splicing, but could also have been achievedas a result of an overall improvement in heme biosynthesis. Indeed, theALAS2 gene, the key regulator of erythroid heme biosynthesis, wasover-expressed compared to that in the mock-treated cells at the samestage of differentiation in V1-treated cells (44% and 74% improvementfor patients A and B, respectively, Table 3). PPIX accumulation wasmonitored during cell differentiation; in cells treated with the V1morpholino, we observed a cumulative reduction of PPIX accumulation aserythroid differentiation progressed, culminating at the late E endpoint stage in a 44% reduction for patient A and a 58% reduction forpatient B, versus their respective mock-treated cells (FIGS. 3A and B).Nevertheless, in overt patient A, PPIX accumulation remained higher thanin the WT IVS3-48T/T control (subject C). Interestingly, PPIXaccumulation decreased in overt EPP patient B to a level similar to thatfound in the asymptomatic patient D (FIG. 3B). This result was inagreement with the total erythrocyte PPIX measured in 40 EPP familiesshowing that the asymptomatic EPP patients accumulated 2.5 times morePPIX than healthy subjects, even though this remained at subclinicallevels (FIG. 4).

This is the first demonstration that the exon 3-4 splicing repair, notonly improves WT FECH mRNA production, but also reduces PPIXaccumulation in erythroid progenitors. Taken together, these resultsshowed that the oligonucleotide-driven shift in splicing from thecryptic exon 3-4 splice site to the WT site, as demonstrated in LBCLsfrom EPP patients, can also occur in primary cultures of erythroidprogenitors, and can increase WT FECH mRNA to such an extent that itdrastically reduces the accumulation of PPIX. It is possible tospeculate that this reduction in PPIX accumulation observed in erythroidcells from an overt patient could reach a level that would besufficiently low to suppress skin sensitivity in vivo.

The correction of FECH exon 3-4 splicing is an attractive therapeuticapproach for EPP, because the IVS3-48C allele is present in more than90% of overt patients. Moreover a modest increase in FECH activity issufficient to shift the patient's status from overt to asymptomatic.Since the correction will occur in bone marrow erythroblasts, willpersist in circulating mature erythrocytes, and since the lifespan ofmature erythrocytes is about 120 days, it is likely that the effects ofthe oligonucleotide treatment will be fairly prolonged. Most EPPpatients present solely non life-threatening dermatological symptoms,and so the use of an integrative gene therapy is not currentlyappropriate. In contrast, antisense therapy has several advantages: i)the splicing correction occurs in the endogenous gene transcribed in itsphysiological environment, preventing over- or inappropriate expression;ii) a pharmalogical treatment is easier to administer than somatic genetherapy; and finally iii) this treatment can easily be simplydiscontinued if adverse effects occur. Targeting pre-mRNA splicing as atherapeutic strategy in Mendelian disorders was proposed several yearsago for Duchenne muscular dystrophy (Wilton et al. 1999), spinalmuscular atrophy (Hua et al. 2008) and β-thalassemia. In 1993, Dominskiet al demonstrated that correct splicing can be restored in vitro by ASOtargeting the β-globin pre-mRNA (Dominski and Kole 1993). Twolaboratories were later able to improve hemoglobin synthesis in vivo,and to reduce cell damage in humanized IVS2-654 thalassemic mice usingeither a morpholino oligomer conjugated to a peptide, or an antisenseRNA vector (Svasti et al. 2009; Xie et al. 2011). The challenge facingantisense therapeutic strategies is to develop efficient ways to targetASO to specific cells, in our case to the erythroid progenitors. Forsystemic administration, it is important to enhance the specificity ofthe treatment while reducing the concentration of ASOs so as to limittoxicity. To enhance ASO targeting, different strategies have beendeveloped, including cell-penetrating peptides, aptamers, and cationicliposome-ASO complexes. For erythropoietic protoporphyria therapy, aninteresting target could be transferrin receptor 1 (CD71), which isexpressed at a very high level during the differentiation stage, whenFECH incorporates iron into PPIX to form heme. Several peptides andaptamers that bind with high affinity to human or mouse CD71 and displayendocytotic properties are already available (Lee et al. 2001; Wilner etal. 2012).

This proof-of-concept of the ability of the V1 antisense morpholinooligonucleotide to restore correct exon 3-4 splicing of IVS3-48 pre-mRNAassociated with a major increase in WT FECH mRNA production, and finallyto produce a marked reduction of PPIX accumulation in culturederythropoietic cells from EPP patients suggests that this or similarcompounds should be tested in a humanized mouse model of EPP, andpossibly subsequently for treating EPP patients.

TABLE 1 LNA-ASOs used in the orientated strategy  Position SequenceLNA Sequence -45-63 5′ GCAGCCTGAGAAATGTTTT 5′ gcEgcLtgEgaEatPttZt 3′ 3′(SEQ ID NO: 2) -54-74 5′ GAAATGTTTTCTACTCAATAA 5′gEaaZgtZttLtaLtcEatEa 3′ 3′ (SEQ ID NO: 11) -97-116 5′AAAACATTTCTCAGGCTGC 5′ aEa aLa tZt cZc aPg cZg c 3′ 3′ (SEQ ID NO: 12)

TABLE 2 FECH genotypes and phenotypes in 3 EPP patients and one controlfrom whom the erythroid precursors were extracted. FECH IVS3-48 TotalErythrocyte FECH Patient mutation genotype Protoporphyrins activityPhenotype A c.1038 T > G C/T 43 1.8 symptomatic p.Y346X B c.1078-2 A > GC/T 45 1.8 symptomatic C ND T/T 2.5 1.7 asymptomatic D WT T/T 1.5 5.6asymptomatic

TABLE 3 WT FECH and ALAS2 mRNA quantifications during in vitroerythropoiesis experiments. ALAS2 mRNA WT FECH mRNA Experiment 1 PatientA mock 0.25 0.29 V1 0.36 0.46 Control subject C mock 1 1 V1 0.75 0.73Experiment 2 Patient B mock 0.86 0.96 V1 1.5 1.9 Patient D mock 1 1 V11.14 0.85

TABLE 4 Oligonucleotides used in RT-PCR and RT-qPCR.Sense primer (5′-3′) Antisense primer (5′-3′) FECH Exon3-4 Cos7TGGACCGAGACCTCATGACA AGTCCATATCTTGATGGGGGA cells (SEQ ID NO: 13)T (SEQ ID NO: 19) FECH Exon 3-4 TAAACATGGGAGGCCCTGAAACGGGTTCGGCGTTTGGCGATGA LBLC (SEQ ID NO: 14) ATGG (SEQ ID NO: 20)WT FECH mRNA TTCCTATTCAGAATAAGCTGGCA GCCTCCAATCCTGCGGTACTG qPCRCCAT (SEQ ID NO: 15) (SEQ ID NO: 21) ALAS2 mRNA qPCRAGGATGTGTCCGTCTGGTGTA TGAAACTTACTGGTGCCTGAG (SEQ ID NO: 16)A (SEQ ID NO: 22) B2M TGCTGTCTCCATGTTTGATGTATC TCTCTGCTCCCCACCTCTAAGTT (SEQ ID NO: 17) (SEQ ID NO: 23) HPRT1 TGACACTGGCAAAACAATGCAGGTCCTTTTCACCAGCAAGCT (SEQ ID NO: 18) (SEQ ID NO: 24)

TABLE 5 LNA-ASOs used in the intron 3 walking. Initial walk in Intron 3SEQ ID Position LNA sequence Native sequence NO:   -1  -15 5′ctEaatcEtttaEca 3 ctaaatcatttaaca 25  -11  -25 5′ taEcatEcaggtEag 3′taacatacaggtaag 26  -21  -35 5′ gtEagtPgatttZat 3′ gtaagtggattttat 27 -31  -45 5′ ttZattLcagctZag 3′ tttattccagcttag 28  -41  -55 5′ctZagcEgcctgEga 3′ cttagcagcctgaga 29  -51  -65 5′ tgEgaaEtgtttZct 3′tgagaaatgttttct 30  -61  -75 5′ ttZctaLtcaatEaa 3′ tttctactcaataaa 31 -71  -85 5′ atEaaaEagaaaEaa 3′ ataaaaaaagaaaaaa 32  -81  -95 5′aaEaaaPcaaaaZtt 3′ aaaaaagcaaaattt 33  -91 -105 5′ aaZtttaPagagLct 3′aattttagagagcct 34 -101 -115 5′ agLctaEcaagaZta 3′ agcctaacaagatta 35-111 -125 5′ gaZtaaPcctttEaa 3′ gattaagcctttaaa 36 -121 -135 5′ttEaaaLagaagLtt 3′ ttaaaacagaagctt 37  -45 -63 5′ gcEgcLtgEgaEatPttZt 3′gcagcctgagaaatgtttt  2 microwalk around position -45 Position sequence-31 -45 5′ ttZattLcagctZag 3′ tttattccagcttag 38 -32 -46 5′ttEttcLagcttEgc 3′ ttattccagcttagc 39 -33 -47 5′ taZtccEgcttaPca 3′tattccagcttagca 40 -34 -48 5′ atZccaPcttagLag 3′ attccagcttagcag 41-35 -49 5′ ttLcagLttagcEgc 3′ ttccagcttagcagc 42 -36 -50 5′tcLagcZtagcaPcc 3′ tccagcttagcagcc 43 -37 -51 5′ ccEgctZagcagLct 3′ccagcttagcagcct 44 -38 -52 5′ caPcttEgcagcLtg 3′ cagcttagcagcctg 45-39 -53 5′ agLttaPcagccZga 3′ agcttagcagcctga 46 -45 -59 5′gcEcccZgagaaEtg 3′ gcaccctgagaaatg 47 -46 -60 5′ caLcctPagaaaZgt 3′caccctgagaaatgt 48 -47 -61 5′ acLctgEgaaatPtt 3′ accctgagaaatgtt 49-48 -62 5′ ccLtgaPaaatgZtt 3′ ccctgagaaatgttt 50 -49 -63 5′ccZgagEaatgtZtt 3′ cctgagaaatgtttt 51 -50 -64 5′ ctPagaEatgttZtc 3′ctgagaaatgttttc 52 -51 -65 5′ tgEgaaEtgtttZct 3′ tgagaaatgttttct 53-52 -66 5′ gaPaaaZgttttLta 3′ gagaaatgttttcta 54 -53 -67 5′agEaatPttttcZac 3′ agaaatgttttctac 55 -45 -63 5′ gcEgcLtgEgaEatPttZt 3′gcagcctgagaaatgtttt  2 microwalk around position -45-63 Positionsequence -43 -63 5′ tagcEgcLtgEgaEatPttZt 3′ tagcagcctgagaaatgtttt 33-44 -63 5′ agcEgcLtgEgaEatPttZt 3′ agcagcctgagaaatgtttt  4 -45 -63 5′gcEgcLtgEgaEatPttZt 3′ gcagcctgagaaatgtttt  2 -43 -64 5′tagcEgcLtgEgaEatPttZtc 3′ tagcagcctgagaaatgttttc  5 t -44 -64 5′agcEgcLtgEgaEatPttZtc 3′ agcagcctgagaaatgttttc  7 -45 -64 5′gcEgcLtgEgaEatPttZtc 3′ tagcagcctgagaaatgtttt 56 -43 -65 5′tagcEgcLtgEgaEatPttZtct 3′ agcagcctgagaaatgtttt 57 -44 -65 5′agcEgcLtgEgaEatPttZtct 3′ agcagcctgagaaatgttttc  9 -45 -65 5′gcEgcLtgEgaEatPttZtct 3′ gcagcctgagaaatgattct 10

For each ASO in intron 3, the position corresponds to the distance ofthe 5′ and 3′ nucleotide from the first exon 4 base. The sequences areshown on the complementary strand. LNA bases are “E” for Adenine, “P”for Guanine, “Z” for Thymine and “L” for Cytosine.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

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The invention claimed is:
 1. A composition comprising one or morestabilized antisense oligonucleotides (ASO), each of which comprises orconsists of a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, wherein stabilization ofsaid one or more stabilized ASO is provided by a chemical modificationselected from the group consisting of a backbone modification,heterocycle modification, sugar modification, conjugation to a peptide,conjugation to aptamer, conjugation to antibody and complex tonanoparticle.
 2. The composition of claim 1, wherein said chemicalmodification is a backbone modification.
 3. The composition of claim 2,wherein said backbone modification is at least one selected from thegroup consisting of methylphosphonate, methylphosphorothioate,phosphorodithioate, and p-ethoxy modification.
 4. The composition ofclaim 1, wherein said one or more stabilized ASO are selected from thegroup consisting of Locked Nucleic Acid (LNA) oligonucleotides,morpholino oligonucleotides, tricyclo-DNA-antisense oligonucleotides andconjugate products thereof.